Staphylococcus aureus is a human pathogen that causes a wide range of diseases from skin and soft tissue infections (SSTI) to life threatening sepsis and pneumonia. Infections with S. aureus in the hospitals and community have been on the rise over the past two decades and the rapid spread of S. aureus strains resistant to methicilin (MRSA) and the last resort antibiotic vancomycin (VISA and VRSA) is posing a major threat to global public health. There are currently no vaccines or immunotherapeutics available for prophylaxis or treatment of S. aureus infections. The pathogenicity of S. aureus is dependent on many virulence factors, including cell surface proteins and polysaccharides, as well as secreted toxins that cause tissue damage, promote bacterial dissemination and metastatic growth in distant organs, and enable the pathogen to evade the host innate immune response. Several vaccine or therapeutic candidates that target individual virulence factors have been tested with little success, highlighting the need for a multi-faceted approach. This is a collaborative proposal relying on S. aureus antibodies developed at Integrated BioTherapeutics (IBT) and a novel technology for generation of multispecific antibodies (Zybodies(tm)) developed by Zyngenia, Inc. A Zybody comprises a conventional monoclonal antibody (mAb) to which peptides (Molecular Recognition Domains, MRDs) are genetically fused to the N- and/or C-termini of the antibody's heavy and light chains. The resulting Zybody bivalently binds the target of the parental mAb and is armed to bind up to 4 additional targets through the incorporated MRDs. The goal of this Phase I SBIR is to employ the Zybody technology for simultaneous targeting of surface polysaccharides and secreted toxins enabling the sequestration of toxins at site of infection, prevention of bacterial dissemination and toxemia, and efficient clearance of th bacteria and toxins by neutrophils. The proposal has two Specific Aims. In Aim 1, Tri-specific Zybodies will be constructed each of which will target one cell-associated virulence factor (capsule polysaccharide Type 8 or wall teichoic acid) along with two soluble toxins (alpha hemolysin and Panton-Valentine leukocidin). Tri-specific Zybodies will be produced and functionally characterized in vitro for their ability to bind and neutralize toxin(s) and enhance S aureus opsonophagocytosis. In Aim 2 the tri-specific zybodies will be tested in several animal models including bacteremia, pneumonia and skin infection models. Efficacy of the candidates will be determined using survival, bacterial burden, and toxemia as endpoints. This Phase I SBIR serves as proof of concept for future development of effective multispecific preclinical candidates for human use. In a subsequent Phase II we envision humanization of the Zybodies, optimization of the MRD sequences, expansion of molecular targets, as well as standard preclinical studies required for submission of an Investigational New Drug Application to FDA.